1. Field of the Invention
This invention relates to the use of transforming growth factor-beta to induce bone growth in vivo and to devices for implantation into a bony site that are treated with transforming growth factor-beta, as well as to pharmaceutical compositions for this purpose.
2. Description of Related Art
The disorders associated with bone loss present major public health problems for Western societies. Osteoporosis alone may affect 20 million Americans in the early years of the next century. Hence, there is wide interest in identifying factors or potential therapeutic agents that inhibit bone loss and stimulate the formation of healthy new bone.
Bone is an extremely complex, but highly organized, connective tissue that is continuously remodeled during the life of an adult by cellular events that initially break it down (osteoclastic resorption) and then rebuild it (osteoblastic formation). This remodeling process occurs in discrete packets throughout the skeleton, i.e., in both cortical bone and trabecular bone. It has recently been reported that mouse bone marrow cells can be stimulated to generate osteoclasts in the presence of parathyroid hormone-related protein or vitamin D. See Akatsu et al., Endocrinology, 125:20-27 (1989); Takahashi et al., Endocrinology 123:2600-2602 (1988) and Takahashi et al., Endocrinology, 123: 1504-1510 (1988).
The currently available therapeutic agents known to stimulate bone formation are fluoride, estrogen, metabolites, and vitamin D. Fluoride clearly increases trabecular bone mass, but questions remain about the quality of the new bone formed, the side effects observed in some patients, whether there are beneficial effects on vertebral fracture rates, and whether increased fragility of cortical bone with subsequent propensity to hip fracture follows.
Another approach is using agents that promote resorption (parathyroid hormone) and then interrupt resorption (calcitonin). One proposed, but not validated, such sequential therapeutic regimen is coherence therapy, where bone metabolic units are activated by oral phosphate administration and then resorption is inhibited by either diphosphonates or calcitonin.
Within the past few years several factors that stimulate osteoblasts were identified in bone, including TGF-.beta., fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor I, and .beta.2 macroglobulin. Of these, TGF-.beta. and IGF-I were deemed attractive candidates for factors linking previous bone resorption with subsequent bone formation. Mundy, The Journal of NIH Research, 1:65-68 (1989).
Other proteins stored in the bone matrix may also be important for bone formation. When demineralized bone was injected into the muscle or subcutaneous tissue of rats, a cascade of events, including chondrogenesis, ensued. Urist, Science, 150:893 (1965). This observed activity was due to bone morphogenetic protein (BMP). Since the 1960s several investigators have attempted to identify and characterize this activity. Thus, a protein of 22 Kd, called osteogenin, was identified that possessed the activity. Sampath et al., Proc Natl. Acad. Sci. USA, 84:7109 (1987). Recently, a glycoprotein called osteoinductive factor was purified that exhibited many of the same properties as TGF-.beta. in vitro but which, unlike TGF-.beta., could induce all of the events involved in the osteoinductive process in vivo. Bentz et al., J. Cell. Biol., 107: 162a (1989). Additionally, three proteins from demineralized ovine bone matrix were identified as having this activity. Wang et al., Proc. Natl. Acad. Sci., 85:9484 (1988) and Wozney et al., Science, 242:1528 (1988). These proteins were named BMP-1, BMP-2A, and BMP-3, the latter two of which belong to the extended TGF-.beta. family by limited sequence homology. These workers modified the assay for bone induction to show cartilage formation but did not show that the proteins ultimately stimulate formation of bone.
The transforming growth factor-beta (TGF-.beta.) group of molecules are each dimers containing two identical polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 Kd. Biologically active TGF-.beta. has been defined as a molecule capable of inducing anchorage independent growth of target cell lines or rat fibroblasts in in vitro cell culture, when added together with EGF or TGF-.alpha. as a co-factor. TGF-.beta. is secreted by virtually all cell types in an inactive form. This latent form can be activated by proteolytic cleavage of mature TGF-.beta. from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-.beta. with the precursor remainder or with a protein binding to TGF-.beta. or with alpha.sub.2 -macroglobulin. This complex is disrupted so as to activate the TGF-.beta. either by exposure to transient acidification or by the action of exogenous proteases such as plasmin or plasminogen activator.
There are at least five forms of TGF-.beta. currently identified, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5. Suitable methods are known for purifying this family of TGF-.beta.s from various species such as human, mouse, green monkey, pig, bovine, chick, and frog, and from various body sources such as bone, platelets, or placenta, for producing it in recombinant cell culture, and for determining its activity See, for example, R. Derynck et al., Nature 316:701-705 (1985); European Pat. Pub. Nos. 200,341 published Dec. 10, 1986, 169,016 published Jan. 22, 1986, 268,561 published May 25, 1988, and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Seyedin et al, J. Biol. Chem., 262:1946-1949 (1987); Cheifetz et al, Cell, 48:409-415 (1987); Jakowlew et al., Molecular Endocrin., 2:747-755 (1988); Dijke et al., Proc. Natl. Acad. Sci. (U.S.A.), 85:4715-4719 (1988); Derynck et al., J. Biol. Chem., 261:4377-4379 (1986); Sharples et al., DNA, 6:239-244 (1987); Derynck et al., Nucl. Acids. Res., 15:3188-3189 (1987); Derynck et al., Nucl. Acids. Res., 15:3187 (1987); Derynck et al., EMBO J., 7:3737-3743 (1988)); Seyedin et al., J. Biol. Chem., 261:5693-5695 (1986); Madisen et al., DNA, 7:1-8 (1988); and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A.), 85:79-82 (1988), the entire contents of these publications being expressly incorporated by reference.
TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5, which are the most recently discovered forms of TGF-.beta., were identified by screening cDNA libraries. None of these three putative proteins has been isolated from natural sources, although Northern blots demonstrate expression of the corresponding mRNAs. TGF-.beta.4 and TGF-.beta.5 were cloned from a chicken chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol., 2:1186-1195 (1988)) and from a frog oocyte cDNA library, respectively. The frog oocyte cDNA library can be screened using a probe derived from one or more sequences of another type of TGF-.beta.. TGF-.beta.4 mRNA is detectable in chick embryo chondrocytes, but is far less abundant than TGF-.beta.3 mRNA in developing embryos or in chick embryo fibroblasts. TGF-.beta.5 mRNA is expressed in frog embryos beyond the neurula state and in Xenopus tadpole (XTC) cells.
TGF-.beta. has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. TGF-.beta. is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science, 233:532 [1986]). For a general review of TGF-.beta. and its actions, see Sporn et al., J. Cell Biol., 105:1039-1045 (1987) and Sporn and Roberts, Nature, 332:217-219 (1988).
The multifunctional activity of TGF-.beta. is modulated by the influence of other growth factors present together with the TGF-.beta.. TGF-.beta. can function as either an inhibitor or an enhancer of anchorage-independent growth, depending on the particular set of growth factors, e.g., EGF or TGF-.alpha., operant in the cell together with TGF-.beta. (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82:119 [1985]). TGF-.beta. also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthritis and Rheumatism, 26:1370 [1983]).
Although TGF-.beta. has been purified from several tissues and cell types, as indicated above, it is especially abundant in bones (Hauschka et al., J. Biol. Chem., 261:12665 (1986)) and platelets (Assoian et al., J. Biol. Chem., 258:7155 (1983)). TGF-.beta. is postulated to be one of the local mediators of bone generation and resorption, because of its presence in large amounts in bone and cartilage, because cells with osteoblast and chondrocyte lineage increase replication after exposure to TGF-.beta., and because TGF-.beta. regulates differentiation of skeletal precursor cells. See Centrella et al., Fed. Proc. J., 2:3066-3073 (1988).
Immunohistochemical studies have shown that TGF-.beta. is involved in the formation of the axial skeleton of the mouse embryo. TGF-.beta. is also present in other embryos in the cytoplasm of osteoblasts in centers of endochondral ossification and in areas of intramembranous ossification of flat bones, such as the calvarium. Heine et al., J. Cell. Biol., 105:2861-2876 (1987). Following in situ hybridization of TGF-.beta.1 probes, localization of TGF-.beta. in both osteoclasts and osteoblasts has been described in development of human long bones and calvarial bones. Sandberg et al., Development, 102:461-470 (1988), Sandberg et al., Devel. Biol., 130:324-334 (1988). TGF-.beta. is found in adult bone matrix (Seyedin et al., Proc. Natl. Acad. Sci. USA. 82:2267-2271 (1985), Seyedin et al., J. Biol. Chem., 261:5693-5695 (1986)) and appears at the time of endochondral ossification in an in vivo model of bone formation (Carrington et al., J. Cell. Biol., 107:1969-1975 (1988)). Cultured fetal bovine bone osteoblasts as well as rat osteosarcoma cells have high mRNA levels for TGF-.beta. and secrete relatively high concentrations of TGF-.beta. (Robey et al., J. Cell. Biol., 105:457-463 (1987)).
In certain in vitro models, TGF-.beta. was found to stimulate the synthesis of collagen, osteopontin, osteonectin, and alkaline phosphatase, and to stimulate replication in osteoblast-like cells. See Centrella et al., J. Biol. Chem., 262:2869-2874 (1987), Noda et al., J. Biol. Chem., 263:13916 (1988), Wrana et al., J. Cell. Biol., 106:915 (1988), Noda et al., J. Cell. Physiol., 133:426 (1987), Pfeilshifter et al., Endocrinology, 121:212 (1987), Centrella et al., Endocrinology, 119:2306 (1986), and Roby et al., J. Cell. Biol., 105:457 (1987). In other in vitro models, TGF-.beta. was found to inhibit proliferation and expression of alkaline phosphatase and osteocalcin. See Centrella et al., supra, Noda and Rodan, Biochem. Biophys. Res. Commun., 140: 56 (1986), and Noda, Endocrinology, 124:612 (1989).
Further, while Centrella et al., supra, showed increased collagen synthesis after treatment of osteoblasts from rat calvaria with TGF-.beta., Robey et al., supra, could not show increased synthesis of collagen in fetal bovine bone osteoblasts, postulating that the increased collagen production is secondary to the effects of TGF-.beta. on the proliferation of osteoblasts. In organ culture, TGF-.beta. was reported to stimulate bone resorption in neonatal mouse calvarias, but inhibit resorption in the fetal rat long bone system. See Tashjian et al., Proc. Natl. Acad. Sci. USA, 82:4535 (1981) and Pfeilshifter et al., J. Clin. Invest., 82:680 (1988). TGF-.beta. activity was reported to be increased in cultures of fetal rat calvaria and in calvarial cells incubated with stimulators of bone resorption, such as parathyroid hormone, 1,25-dihydroxyvitamin Ds, and IL-1 (Petkovich et al., J. Biol. Chem., 262:13424-13428 (1987), Pfeilschifter and Mundy, Proc. Natl. Acad. Sci. USA, 84: 2024-2028 (1987)). Furthermore, it was reported that TGF-.beta. inhibits the formation of osteoclasts in bone marrow cultures. Chenu et al., Proc. Natl. Acad. Sci. USA, 85:5683-5687 (1988). The showing that TGF-.beta. has effects on both osteoclasts and osteoblasts led Pfeilschifter and Mundy, supra, to propose that it is involved in the strict coupling of the processes of bone resorption and bone formation characteristic of the remodeling process in adult bone. It has also been postulated that the local acidic, proteolytic environment provided by the osteoclasts results in activation of matrix-associated latent TGF-.beta.. Oreffo et al., Calcified Tiss. Internatl., 42: Suppl:A15 (1988).
In view of the conflicting results reported for in vitro activities, it is not clear whether in vitro models can be used to predict the effects of TGF-.beta. on bone formation and resorption in vivo. See Roberts et al., Proc. Natl. Acad. Sci. USA, 82:119 (1985).
Additional references reporting that TGF-.beta. promotes the proliferation of connective and soft tissue for wound healing applications include U.S. Pat. No. 4,810,691 issued Mar. 7, 1989, U.S. Pat. No. 4,774,228 issued Sep. 27, 1988, Ignotz et al., J. Biol. Chem., 261:4337 [1986]; J. Varga et al., B.B.Res.Comm., 138:974 [1986]; A. Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 78:5339 [1981]; A. Roberts et al., Fed. Proc., 42:2621 [1983]; and U.S. Pat. No. 4,774,228 to Seyedin et al. TGF-.beta. stimulates the proliferation of epithelia (T. Matsui et al., Proc. Natl. Acad. Sci. U.S.A., 83:2438 [1986]; G. Shipley et al. Cancer Res., 46:2068 [1986]); induces collagen secretion in human fibroblast cultures (Chua et al., J. Biol. Chem., 260:5213-5216 [1983]); stimulates the release of prostaglandins and mobilization of calcium (A. Tashjian et al., Proc. Natl. Acad. Sci. U.S.A., 82:4535 [1985]); and inhibits endothelial regeneration (R. Heimark et al., Science, 233:1078 [1986]).
In wound chambers implanted subcutaneously, TGF-.beta. increased DNA and collagen production. Sporn et al., Science, 219:1329 (1983) and Sprugel et al., Am. J. Pathol., 129:601 (1987). Moreover, TGF-.beta. produced collagen fibrosis when injected subcutaneously (Roberts et al., Proc. Natl Acad. Sci. USA, 83:4167-4171 (1986)) and promoted healing of skin incisions in rats (Mutoe et al., Science, 237:1333 (1987)). Nevertheless, although TGF-.beta. induced chondrogenesis in muscle-derived cells in vitro (Seyedin et al., Proc. Natl. Acad. Sci. USA, 82:2267 (1985) and Seyedin et al., J. Biol. Chem., 261:5693 (1986)), it did not produce cartilage in vivo even when implanted with collagenous substrates, a system used for a long time as a bone induction model in animals (Sampath et al., Proc. Natl. Acad. Sci. USA, 84:7109 (1987) and Howes et al., Calcif. Tissue Int., 42:34 (1988)).
New studies have shown a time-dependent appearance of mRNA for TGF-.beta.1 at a fracture site in a rat and have localized the peptide immunohistochemically in the periosteum of the healing fracture; the same researchers reported that injections of TGF-.beta.1 into the periosteal area of the femur of young rats have caused significant formation of new cartilage. Bolander et al., New York Academy of Sciences, "Transforming Growth Factor-.beta.s: Chemistry, Biology and Therapeutics, May 18-20, 1989. It has been found that injections of TGF-.beta.1 into the parietal bone of young rats stimulated periosteal bone formation, resulting in a thickening of the calvarium. Noda et al., J. Cell. Biol., 107:48 (1988). TGF-.beta. was reported to stimulate local periosteal woven bone formation when injected daily onto the periostea of parietal bones of neonatal rats. Noda and Camilliere, Endocrinology, 124:2991-2994 (1989).
Certain researchers have found that TGF-.beta. does not induce bone formation unless it is administered concurrently with a cofactor, e.g., an osteoinductive factor purified from bovine demineralized bone. Bentz et al., supra, U.S. Pat. No. 4,843,063 issued Jun. 27, 1989 to Seyedin et al., and U.S. Pat. No. 4,774,322 issued Sep. 27, 1988.
The above studies are inconclusive and inconsistent regarding the formation of mature, histologically normal bone with TGF-.beta. alone. For example, the bones being generated by Noda et al., 1989, supra, were neonatal, i.e., not fully formed with large spaces and increased cartilage formation. In addition, woven bone is resorbed before mature bone is laid down. Thus there is still a demonstrated need for a bone induction agent that will induce bone only where it is needed, does not have side effects in some patients as does fluoride treatment, and does not require addition of a cofactor or another peptide growth factor for acceleration of repair of damaged bone in vivo.
Accordingly, it is an object of the present invention to provide exogenous TGF-.beta. to a local site on an animal where skeletal (bony) tissue is deficient without administering a bone-inducing cofactor so as to produce in every case mature, morphologically normal bone at the site of administration where it is needed.
It is another object to provide a device for implantation into an animal for generation of bone that is treated with TGF-.beta. in such a way as to induce bone at the implantation site.
These and other objects will become apparent to those skilled in the art.